The goal of the proposed research is to develop an understanding, at the cellular, subcellular, and molecular levels of the hormonal events relating to calcium metabolism in the kidney. Of particular interest is the regulation of the metabolism of 25-hydroxyvitamin D3 (25-OH-D3) to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the potent biologically active hormonal form of vitamin D and 24,25-dihydroxyvitamin D3, the biological activity of which remains an unresolved puzzle. Specifically, it is proposed to: (1) Utilize primary cultures of chick kidney cells to continue to elucidate the actions, interactions, and mechanisms of actions, of various regulators of 25-OH-D3 metabolism. These include parathyroid hormone (PTH), 1,25(OH)2D3, steroid hormones such as glucocorticoids and sex steroids, calcium and phosphate. Radioactively labeled 25-OH-D3 will be incubated with cultures following various treatments and separated from its metabolites by high performance liquid chromatography. (2) Measure changes in circulating PTH levels in the chick in vivo in response to various signals by using a newly developed highly sensitive bioassay based on the cyclic AMP generated in cultured chick kidney cells. (3) Pacify the cytochrome P450 responsible for 1-hydroxylase activity from the kidney mitochondria of vitamin D-deficient chicks and produce both polyclonal and monoclonal antibodies to the protein. These will be used to probe changes in the levels of this protein in response to the various regulatory signals listed above as well as to determine whether 1-hydroxylation and 24-hydroxylation of 25-OH-D3 are catalyzed by the same protein. (4) Determine whether the phospholipid composition of the chick kidney mitochondria is correlated with or has an influence on 25-OH-D3 metabolism. Although the regulation of the metabolism of 25-OH-D3 has been under investigation for a number of years, little is understood regarding the subcellular details of this complex process. The proposed work will contribute not only to a deeper understanding of the clinically important calcium regulating hormones but will also contribute conceptually to the understanding of other equally important steroid hormone generating systems.